Sensory neurons express a multiple array of membrane receptors reacting to neurotrophin, peptides, and pain mediators, leading thus to an intrinsic heterogeneous population. Among pain receptors expressed by neurons responsive to NGF, some of them are powerfully modulated by it such as the ones expressing nociceptive transient receptor potential vanilloid 1 (TRPV1) receptors (Wang and Woolf, 2005). In fact, on trigeminal neurons, some nociceptors that do not express TRPV1 receptors express purinergic P2X3 ones (Simonetti et al., 2006). In this work, we wondered whether the action of NGF might also involve other receptors such as ATP-sensitive P2X3 receptors, which are important transducers of nociceptive stimuli on sensory neurons (Cockayne et al., 2000, 2005; Souslova et al., 2000). P2X3 receptors of sensory neurons are a major target for the action of several proalgesic substances (North, 2004).

We report that neutralization of endogenous NGF decreased P2X3 receptor-mediated currents and delayed their recovery from desensitization via PKC-dependent threonine phosphorylation. This treatment also favored the expression of the splicing P2X2 variant P2X2e, which could contribute to heteromeric P2X2/3 receptors (Koshimizu et al., 2006). We suggest that such changes might transform the ability of TG neurons to react to ATP-dependent noxious stimuli, a phenomenon we also investigated in vivo.

Unless otherwise stated, the standard protocol for manipulation of NGF levels included 24 h treatment of cultures with either the anti-NGF antibody (1:5000) or NGF (50 ng/ml) and washout with control medium when tests of cultured cells for molecular biology or electrophysiological or imaging experiments commenced. In separate experiments aimed at testing acute effects of NGF or its antibody, these substances were applied for 6–15 min via fast superfusion.

All tests were completed within 40–60 min to avoid recovery from either treatment. A scatter plot analysis of P2X3 receptor-mediated current amplitude versus time within this time frame indicated no significant time-dependent alteration during the washout phase (the correlation coefficient was r = −0.15 for anti-NGF antibody, p = 0.8; and r = −0.12, p = 0.5 for NGF treatment, respectively).

The efficacy of the neutralizing activity of the anti-NGF antibody (Sigma) was demonstrated by Ro et al. (1999) and further validated in our laboratory. For this purpose, PC12 cells (10 d in culture) grown with NGF (50 ng/ml) in the presence of the anti-NGF antibody (6 μg/ml) displayed minimal neuronal processes (<10% of cells had processes 13 ± 5 μm long), unlike NGF-treated controls (85% of cells had processes that were 50 ± 37 μm long; n = 210) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). To exclude cross-reactivity of anti-NGF antibodies with other neurotrophins, the dot blot of NGF, BDNF, glial cell line-derived neurotrophic factor, and neurotrophins 3 and 4 (50 ng; Alomone Labs or Sigma) was immunostained with anti-NGF antibody (1:2000; Sigma), showing staining for NGF only and nonspecific signals.

Real-time reverse transcription-PCR.

Reverse transcription (RT) from total mRNA of TG cultures was performed as reported previously (Simonetti et al., 2006). Real-time PCRs were run in duplicate in a Bio-Rad (Hercules, CA) iQ5 thermocycler using IQ SyBr Green Supermix. Reactions were performed in the presence of specific primers to measure the presence and the relative abundance of mouse P2X3 or P2X2 (supplemental Table 1, available at www.jneurosci.org as supplemental material). Primers to amplify specific splicing variants were designed to overlap the splicing boundaries. Data normalization was carried with respect to neuronal β-tubulin III and glyceraldehyde-3-phosphate dehydrogenase housekeeping mRNA content. Calculations for relative mRNA transcript levels were performed using the comparative method between cycle thresholds of different reactions (Simonetti et al., 2006). Experimental treatments did not affect the expression of the housekeeping genes chosen. Negative controls containing no template cDNA were run with each pair of primers in each condition and gave no result. PCR amplifications of TG cultures lacking neurons (Simonetti et al., 2006) or performed without reverse transcriptase reaction did not produce any signal. End-point PCR amplicons of P2X2 variants were separated on agarose gels, purified, and sequenced using BigDye Terminator version 3.0 Ready Reaction Cycle Sequencing kit (Applied Biosystems, Foster City, CA). The sequences were analyzed with the ABI PRISM 3700 DNA Analyzer (Applied Biosystems), showing the expected nucleotide sequence (Koshimizu et al., 2006).

Patch-clamp recording.

After 1 d in culture, cells were continuously superfused (2 ml/min) with physiological solution containing the following (in mm): 152 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH, as previously reported (Fabbretti et al., 2006). Cells were voltage clamped (whole-cell configuration) using pipettes filled with the following (in mm): 140 KCl, 0.5 CaCl2, 2 MgCl2, 2 Mg2ATP3, 2 GTP, 10 HEPES, and 10 EGTA, pH adjusted to 7.2 with KOH (at −60 mV, 70% series resistance compensation). Agonists were applied with a fast superfusion system (Rapid Solution Changer RSC-200; Bio-Logic, Claix, France) with solution exchange time (10–90%) of 30–40 ms. Responses to agonists were measured in terms of peak amplitude. To express agonist potency in terms of EC50 values (concentration producing 50% of the maximum response), dose–response curves for the selective P2X3 receptor agonist α,β-methyleneATP (α,β-meATP) were constructed by applying different agonist doses to the same cells, normalizing data with respect to the effect evoked by 10 μm concentration, and fitting them with a logistic equation (Origin 6.0; Microcal, Northampton, MA). Data validation by fitting the same data points with the Boltzmann equation produced the same results in terms of calculating EC50 values (see Fig. 1B). For standard tests of cell responsiveness, agonist applications (10 μm, 2 s; fast superfusion) were spaced at 5 min intervals to obtain full response recovery from desensitization. The onset of desensitization was estimated by calculating the first time constant of current decay (τfast) in accordance with our previous reports (Fabbretti et al., 2006; Simonetti et al., 2006). Recovery from desensitization was assessed by paired-pulse experiments (Fabbretti et al., 2006). The kinetics of P2X2/3 current decay in heterologous expression systems are primarily influenced by the relative ratio between P2X3 and P2X2 injected cDNAs (Liu et al., 2001). Native P2X2/3 receptors display biphasic currents that reach steady-state conditions at the end of agonist application (Burgard et al., 1999; Grubb and Evans, 1999; Simonetti et al., 2006). We assumed that currents comprising a residual current at the end of a 2-s-long agonist application (Iresidual) ≥5% of peak current (Ipeak) were suggestive of heteromeric P2X2/3 receptors (Grubb and Evans, 1999; Simonetti et al., 2006). This calculation was used to normalize the slow current to the size of the preceding fast response that could vary depending on the experimental protocol.

Capsaicin was applied at the standard test dose of 1 μm (2 s) to evoke reproducible inward currents. Recording of functional responses started ∼10 min after the washout of the culture medium. To minimize data variability, data from experiments based on 24 h treatments were always compared with responses from sister control dishes maintained in vitro for equivalent time on the same dates. For acute treatments, the same cells served as control by comparing responses before and after application of the test agent.

Ca2+ imaging.

Cells were incubated for 40 min at 20–22°C in physiological solution containing Fluo3-AM (5 μm; Invitrogen) and then washed for 30 min. Fluorescence emission was acquired with a CCD camera (Coolsnap HQ; Roper Scientific, Duluth, GA) at 150 ms intervals. Data were collected from cells that produced a rapid response to a pulse of KCl (50 mm, 1 s), thus indicating their neuronal nature. Analysis was performed with the Metafluor software (Metafluor Imaging Series 6.0; Molecular Devices). Intracellular Ca2+ transients were expressed as percentage amplitude changes with respect to baseline.

Behavioral tests for trigeminal pain.

To ascertain whether systemic injection of the anti-NGF antibody into mice could modify subsequent pain responses mediated by activation of P2X3 subunit-containing receptors of trigeminal sensory neurons, we used a modified method (Luccarini et al., 2006) that relies on the nociceptive stimuli applied to the upper lip to evoke a transient painful response lasting 30–45 min. To obtain selective activation of P2X3 receptors, we locally injected α,β-meATP. Because of the invasive nature of these experiments, the minimal number of animals suitable to reach statistically valid data were used. These procedures were approved by the local ethical committee for animal experimentation and run in the animal facility of the University of Trieste. All experiments were in accordance with the guidelines of the International Association for the Study of Pain. In detail, male postnatal day 25–32 C57BL mice (12–18 g body weight) were maintained in a temperature-controlled room at 22°C (12 h alternating light and dark cycle) and were given food and water ad libitum. For testing pain responses, individual mice were placed into a plastic cage with a transparent bottom surface through which behavior was recorded with a video camera. Mice were randomly assigned to four groups (six to eight per group) and received a single intraperitoneal injection of either anti-NGF [2.5 S-NGF (Sigma), 300 ng/g body weight (Banik et al., 2005)] or saline (10 μl/g) 24 h before testing nociceptive responses. On the day of testing, each animal was first placed inside the test box for a 5 min adaptation period to minimize stress. To test nociceptive responses, pretreated mice were injected with 10 μl of α,β-meATP [10 mm (Shinoda et al., 2005)] or saline solution. Injections were made through a 27 gauge needle into the upper lip just lateral to the nose. After injection, mice were immediately placed back inside the test box for a 30 min observation period. Nociception was scored as time (in seconds) spent rubbing the injected area with the forepaws (Luccarini et al., 2006) during the first 15 or the second 15 min epoch from the injection.

Data analysis.

Data are expressed as mean ± SEM, and n indicates the number of experiments in molecular biology/immunocytochemistry or the number of investigated cells in electrophysiology/imaging or animals in behavioral test. Statistical analysis was performed using the Student's t test, the Mann–Whitney rank sum test, or the ANOVA test, as appropriate. A p value of <0.05 was accepted as indicative of a statistically significant difference.

The 24 h changes in NGF concentrations did not affect responses evoked by GABA (applied for 2 s at the juxtathreshold concentration of 10 μm or the maximally effective concentration of 100 μm) (supplemental Fig. 2A, available at www.jneurosci.org as supplemental material). Although NGF is known to rapidly upregulate TRPV1 receptors (Bonnington and McNaughton, 2003; Zhang et al., 2005) including those of TG neurons (Simonetti et al., 2006), 24 h manipulation of NGF levels did not significantly alter the average current amplitude evoked by capsaicin (applied for 2 s at the juxtathreshold concentration of 0.1 μm or the near-maximally effective concentration of 1 μm) (supplemental Fig. 2B, available at www.jneurosci.org as supplemental material). Likewise, despite the fact that only a minority of mouse TG neurons in culture express functional TRPV1 receptors (Simonetti et al., 2006), the number of TG neurons sensitive to 1 μm capsaicin (control: 45 of 172, 26%; anti-NGF: 15 of 71, 21%; NGF: 21 of 77, 31%) or immunoreactive for TRPV1 (control, 36 ± 2%; anti-NGF antibody, 35 ± 5%; NGF, 37 ± 3%; n = 5 experiments) was not changed by 24 h exposure to NGF or anti-NGF anti-body. Supplemental Figure 3 (available at www.jneurosci.org as supplemental material) shows that only approximately half of TRPV1-immunoreactive neurons also expressed TrkA immunoreactivity.

Properties of desensitization of P2X3 receptors

P2X3 receptor signaling is limited by desensitization (Cook and McCleskey, 1997; Sokolova et al., 2004, 2006), a process attenuated by algogens (Paukert et al., 2001; Fabbretti et al., 2006). Thus, we tested whether manipulating NGF could also change desensitization onset and/or recovery from it, using a standard protocol based on paired-pulse application of α,β-meATP as exemplified in Figure 2A in which the two paired current responses are superimposed for comparison. After 24 h NGF or anti-NGF antibody treatment, there was no change in the average current decay (indicative of desensitization onset and expressed as τfast) (Fig. 2B). Nevertheless, recovery from desensitization was significantly increased by NGF and decreased by the anti-NGF antibody (Fig. 2C).

Manipulating NGF affects desensitization of currents induced by α,β-meATP. A, Representative examples of currents induced by applications of α,β-meATP (black bar) in control condition (left), after anti-NGF antibody treatment (middle), and after NGF treatment (right). Traces of 10 μm α,β-meATP-evoked currents recorded after a second agonist application (30 s interval; see arrows) are superimposed. Note the differential recovery from desensitization in the three conditions. B, Histograms show no change in the time constant values of the α,β-meATP-evoked current decay, used as an index of desensitization onset (τfast; n = 151, n = 56, and n = 46 for control, anti-NGF, and NGF, respectively). C, Histograms show that anti-NGF treatment reduces recovery (*p < 0.0001) from desensitization of P2X3-mediated currents, whereas NGF increases (*p < 0.001) this value (n = 99, n = 49, and n = 38 for control, anti-NGF, and NGF, respectively). D, Representative traces of Ca2+ transients show that, with paired α,β-meATP application (black dots, 30 s spaced), the second response is more depressed after anti-NGF treatment than in control. Neurons are identified by their responsiveness to KCl (50 mm, 1 s), which remains unchanged after anti-NGF treatment. E, Histograms showing the depressant action of anti-NGF treatment on the amplitude of Ca2+ transients induced by 10 μm α,β-meATP or 50 mm KCl. Control level is indicated by a dashed line. *p ≤ 0.05; n = 4–5 experiments (30–40 cells in each experiment).

Anti-NGF treatment decreases Ca2+ transients evoked by α,β-meATP

To explore how P2X3 receptor activity could be translated into changes in excitability as a consequence of NGF deprivation, single-cell Ca2+ imaging was used to avoid intracellular dialysis caused by whole-cell patch clamping.

Previous data have shown that, among P2X receptors, activation of P2X3 receptors, in particular, is followed by a relatively rapid increase in intracellular Ca2+ presumably via membrane depolarization (Koshimizu et al., 2000). Because TG neurons express a heterogeneous population of voltage-activated Ca2+ channels, of which the high-threshold, transient N-type is the largest component (Kim and Chung, 1999; Ikeda and Matsumoto, 2003), the fast nature of P2X3 receptor activation should be a powerful stimulus to increase intracellular Ca2+.

Figure 2, D and E, shows that, after anti-NGF antibody treatment, the Ca2+ signal amplitude induced by α,β-meATP was small, indicating reduced P2X3 receptor-dependent excitability [compare Ca2+ responses in control (top) and treated (bottom) neurons in the examples of Fig. 2D], whereas responsiveness to a pulse of K+ (50 mm; 1 s; producing a maximal amplitude Ca2+ rise) remained the same (Fig. 2D,E). The unchanged responses to high-K+-induced depolarization indicated that there was no apparent downregulation of voltage-activated Na+ and Ca2+ channels with consequent fall in neuronal excitability detectable only when P2X3 receptor activity was stimulated. Furthermore, after NGF deprivation, strong depression of the amplitude of the second response to α,β-meATP (Fig. 2D) was preserved (as observed also with patch-clamp recording) (Fig. 2A).

To validate these findings of P2X3 subunit phosphorylation with electrophysiological tests, we investigated whether acute application of NGF could also reverse the depressant effect of anti-NGF treatment on P2X3 receptor-mediated currents. On average, the peak amplitude of the current induced by α,β-meATP significantly (p = 0.02) grew with NGF after anti-NGF treatment (181 ± 24%; n = 12 in control and n = 15 in anti-NGF treatment).

NGF controls membrane delivery of heteromeric P2X2/3 receptors

Mixed current responses (comprising an initial peak and a late slow component termed residual current) evoked by α,β-meATP are generated by a minority of control TG neurons (Simonetti et al., 2006). Figure 5A shows that the average amplitude of the residual current was significantly larger after anti-NGF treatment or NGF application. Note, however, that the average amplitude of the residual current did not exceed 40 pA, a value more than one order of magnitude smaller than the peak current (−432 ± 31 pA; n = 104). Figure 5B demonstrates the increased probability of detecting neurons with residual current (as a fraction of peak amplitude; see Materials and Methods) after anti-NGF antibody treatment, although it was the same after chronic NGF application. A residual current during α,β-meATP application (Burgard et al., 1999) might be considered to be attributable to heteromeric assembly of P2X2/3 subunits (Liu et al., 2001; North, 2002). Thus, our observations raised the possibility that the receptor subunit composition had been changed by anti-NGF treatment.

The increased likelihood of detecting residual currents plus enhanced perimembrane expression of P2X2 subunits concurred to suggest that anti-NGF treatment facilitated coassembly of P2X2 and P2X3 subunits at membrane level. This issue was then directly investigated by cross-linking P2X2 and P2X3 subunits with the membrane-impermeable DTSSP, followed by immunopurification of P2X3 subunit-containing membrane receptors. Unlike control conditions, anti-NGF antibody application enabled detection of coprecipitated P2X3 and P2X2 subunits (n = 5 experiments) (Fig. 5E), strongly suggesting the possibility that such a treatment increased the expression of heteromeric P2X2/3 receptors at membrane level. Similar results were also obtained by omitting the membrane cross-linker (supplemental Fig. 4, available at www.jneurosci.org as supplemental material), although in this case, the purification of the P2X2–P2X3 interacting subunits included also the intracellular fraction and were not restricted to membrane receptors only.

In accordance with immunoprecipitation results, coimmunofluorescence experiments using anti-P2X2 and anti-P2X3 antibodies on cultures treated with the anti-NGF antibody (Fig. 5F) also demonstrated that a larger (p = 0.023) fraction of P2X3 receptor-positive neurons (77 ± 2%; n = 4 experiments) was immunoreactive also for P2X2 when compared with control (57 ± 1%; n = 4). Chronic NGF application did not change the number of P2X2-immunoreactive cells (58 ± 1%; n = 4) (Fig. 5F) (supplemental Fig. 5, available at www.jneurosci.org as supplemental material). Size distribution analysis of P2X2-immunoreactive neurons demonstrated that, in the absence of NGF, the expression of P2X2 subunits increased in medium- to large-sized TG neurons, which did not usually show such subunit before (supplemental Fig. 5B,C, available at www.jneurosci.org as supplemental material).

Although we could not exclude the possibility that homomeric P2X2 receptor expression had also been modulated by manipulating NGF concentrations, our tests using α,β-meATP, an agonist highly selective for P2X3 subunits, indicate that the sustained membrane currents recorded after 24 h anti-NGF treatment were indeed attributable to activation of P2X3 subunit-containing receptors. Globally, these data suggested that blocking endogenous NGF enhanced the expression of P2X2 subunits by P2X3-immunoreactive neurons, increasing the probability of their coassembly at membrane level.

NGF controls P2X2 subunit splicing

Despite the increased immunoreactivity and membrane expression of P2X2 subunits, manipulation of NGF did not modify total P2X2 mRNA measured with real-time RT-PCR because there was no fractional change versus control (1.1 ± 0.12 for anti-NGF and 1.2 ± 0.15 for NGF; n = 3). Thus, we investigated whether the different splicing forms of P2X2 mRNA (P2X2a, P2X2b, and P2X2e) (Fig. 6A), recently cloned from mouse pituitary gland (Koshimizu et al., 2006), could be present in mouse TG neurons and contribute to P2X2/3 receptor activity. Figure 6B demonstrated that P2X2e mRNA was actually present in intact ganglion tissue (lane 4) as well as in control culture (lane 1). Lane 2 of Figure 6B shows that all three isoforms were detectable in cultured neurons after anti-NGF treatment when primers common for all subunits were used, even if P2X2b was very low. Amplicons had the expected length (686, 476, and 410 bp for P2X2a, P2X2b, and P2X2e, respectively) and were fully sequenced to confirm their origin. Lane 3 of Figure 6B indicates that, under similar conditions, when a primer selective for the P2X2e splicing variant was used, this generated a clear signal similar to the one found in the ganglion (lane 4). We next examined whether P2X2 mRNA variants might have been differentially modulated by adding NGF. No significant differences were found in both P2X2a and P2X2e mRNA levels after NGF application (1.1 ± 0.2- and 1.3 ± 0.25-fold respectively; n = 3) (Fig. 6C). Conversely, after anti-NGF treatment, the P2X2b and P2X2e splicing forms were significantly increased (1.5 ± 0.3- and 2.1 ± 0.35-fold, respectively; n = 3 experiments; p = 0.04 and p = 0.02) (Fig. 6C), whereas P2X2a was slightly yet significantly downregulated (0.65 ± 0.2-fold; n = 3; p = 0.04) (Fig. 6C).

Because the anti-NGF treatment affected primarily the P2X2e variant, we wondered whether the P2X2e protein was expressed in TG culture. Because of the lack of 90 amino acids in the P2X2e variant and the existence of multiple immature isoforms of P2X2 subunits with different degree of glycosylation (Koshimizu et al., 2006), it was necessary to deglycosylate protein extracts with PNGaseF to detect discrete bands in Western blot analysis. After deglycosylation, Western immunoblots with anti-P2X2 antibody allowed detecting a single band (50 kDa) in total extracts from control cultures (Fig. 6D, lane 1) corresponding to the nonglycosylated P2X2 polypeptide. The same protocol applied to total extracts from anti-NGF-treated cultures revealed a more complex signal spanning from 50 to 45 kDa (n = 4) (Fig. 6D, lane 2), suggestive of multiple P2X2 isoforms. To confirm this possibility, membrane extracts (n = 2) were thus processed with the cross-linker DTSSP, which being membrane impermeable, enabled us to explore the presence of different isoforms within the P2X2/3 heteromeric receptor expressed at membrane level (Fig. 6D, lanes 3–5). Only in anti-NGF-treated samples was it possible to detect distinct deglycosylated forms (lane 5) comprised within the signal band found in total extracts (lane 2). These data suggest that anti-NGF treatment promoted upregulation of a recently discovered P2X2 splice variant that perhaps might facilitate the expression of heteromeric P2X2/3 receptors.

The effect of NGF does not depend on CGRP release

Because we found recently strong P2X3 receptor potentiation after CGRP treatment (Fabbretti et al., 2006), we wondered whether the effects of NGF treatment/depletion observed in the present study might have been mediated by endogenous CGRP release because NGF can stimulate release of this peptide stored in secretory vesicles of TG neurons (Bowles et al., 2004). Hence, we tested whether NGF could retain its potentiating effect on P2X3 receptors when CGRP receptors were blocked by BIBN4096BS, a selective nonpeptide antagonist of CGRP receptors (Olesen et al., 2004).

Figure 7A shows sample traces indicating that, after coapplication of NGF and BIBN4096BS (50 nm) for 24 h, there was as much potentiation (on average to 145 ± 19% of BIBN4096BS alone; n = 27; p = 0.034) of α,β-meATP-induced currents as the one observed in the absence of the CGRP antagonist (152 ± 18% of control; n = 16; p = 0.0033). BIBN4096BS alone did not change (91 ± 11%; n = 26) the effect of α,β-meATP (see also sample records in Fig. 7A). We also checked that BIBN4096BS was an effective antagonist of CGRP: indeed, in the presence of 50 nm BIBN4096BS, 1 h application of 1 μm CGRP lost its ability to enhance the amplitude of α,β-meATP-induced currents (118 ± 30% of the response obtained with BIBN4096BS alone; n = 9). These data indicate that the potentiating action of NGF on P2X3 receptors was not mediated by endogenous CGRP release.

Modulation by anti-NGF treatment of trigeminal pain evoked by α,β-meATP

To understand whether the observed changes in P2X3 subunit-containing receptor activity after 24 h anti-NGF treatment could be detected also in terms of modulation of trigeminal pain evoked by the P2X3 receptor agonist α,β-meATP, we tested trigeminal pain in accordance with the method of Luccarini et al. (2006). This approach relies on nociception induced by acute focal injection of an algogen into the mouse upper lip and measuring the time spent by mice to rub off (with their forepaws) the subsequent irritation. Figure 8A shows that acute saline injection induced an early nociceptive response that waned after 15 min. This behavior was not changed when mice had been pretreated with anti-NGF antibody (24 h) instead of just saline (Fig. 8A). Figure 8B shows that acute injection of α,β-meATP induced a longer-lasting painful responses that was statistically different (p = 0.005) from control. This more persistent nociceptive phase could not be observed when previous treatment with anti-NGF antibody was performed. These data indicate that 24 h anti-NGF treatment blocked the trigeminal pain induced by α,β-meATP.

Trigeminal nociception evoked by α,β-meATP is prevented by anti-NGF treatment. A, Histograms showing the time spent by mice in face-rubbing activity after subcutaneous injection of α,β-meATP (10 μl bolus of 10 mm solution) into the upper lip after pretreatment with anti-NGF or saline (sal). Each bar is the mean of six to eight animals observed for the first 15 min epoch. No significant changes in rubbing activity are observed during this period. All mice were pretreated 24 h earlier with saline (10 μl/g, i.p.) or anti-NGF antibody (300 ng/g, i.p.). B, In the 15–30 min epoch, a significant increase (*p = 0.005) in rubbing activity is observed after injection of α,β-meATP (n = 8). This algogenic effect of α,β-meATP is blocked (*p= 0.025 vs sham pretreatment) after anti-NGF pretreatment (n = 7).

Discussion

The principal finding of this work is the novel demonstration that NGF neutralization by anti-NGF antibody treatment strongly decreased P2X3 receptor activity on nociceptive TG neurons in vitro and in vivo. Our data might help to explain certain mechanisms responsible for the analgesia induced by anti-NGF antibody treatment as proposed for headache syndromes (Saldanha et al., 1999; Sarchielli and Gallai, 2004) on the basis of the involvement of NGF in chronic inflammatory pain (Woolf et al., 1994) and migraine (Sandler, 1995).

Nociceptive receptors as targets to express the algogenic action of NGF

NGF is one important factor to shape the phenotype of nociceptive neurons (Lindsay and Harmar, 1989; Chao, 2003). It is, therefore, no surprise that antibodies to neutralize endogenous NGF have been considered as therapeutic strategies to control pain (Hefti et al., 2006). Nevertheless, the molecular mechanisms underlying changes in nociceptive neurons after such antibody treatment remain unclear.

Previous studies have shown that NGF primarily potentiates responses mediated by the pain receptor TPRV1 on dorsal root ganglion neurons (Shu and Mendell, 1999; Zhang et al., 2005), an effect blocked by anti-NGF treatment (Amaya et al., 2004). As far as TG neurons are concerned, NGF-dependent upregulation of TRPV1 activity does occur, but it appears to be an acute phenomenon (Simonetti et al., 2006). The limited timespan of the NGF-evoked heat hyperalgesia has been confirmed in vivo (Zhuang et al., 2004) and might be partly attributable to tachyphylaxis of TRPV1 receptors, which is only transiently counteracted by NGF (Shu and Mendell, 2001). Furthermore, TRPV1 antagonism decreases, yet it does not abolish, nociceptive responses of rat TG neurons (Varga et al., 2005). In summary, it is likely that sustained pain signaling by mouse TG neurons requires synergy of activity by various pain sensing receptors.

P2X3 receptors as targets for anti-NGF treatment

Neutralization of endogenous NGF with antibodies against NGF decreased the peak amplitude of P2X3 receptor-mediated currents and delayed their recovery from desensitization. These effects thus provide a mechanism for limiting the ability of P2X3 receptors to respond to repeated nociceptive stimuli. The action of anti-NGF treatment was not accompanied by a change in the agonist potency on P2X3 receptors or in the number of immunoreactive neurons. Likewise, there was no comparable increase in P2X3 protein expression. Manipulating NGF levels did not change neuronal currents evoked by GABA or capsaicin.

Immunocytochemical experiments indicated that small- to medium-diameter TG neurons (used for the present study) predominantly expressed NGF TrkA receptors, often colocalized with P2X3 receptors. Nevertheless, because changes in P2X3 receptor function were systematically observed, we cannot exclude that the effects of NGF/anti-NGF treatment were also mediated by paracrine actions affecting a large population of P2X3-expressing neurons. It is noteworthy that TrkA receptors are the principal mediators of the algogenic action of NGF (Chao, 2003), thus suggesting a role for NGF in P2X3 receptor modulation.

The P2X3 receptor possesses a large number of intracellular threonine residues that can be the target of different kinases, including ectokinases (Pawlowska et al., 1993; Wirkner et al., 2005), allowing for a complex modulation of receptor expression and function by NGF. Our present data demonstrating block of NGF effects on P2X3 receptors by the PKC inhibitor chelerythrine further points to a role for this kinase. Although recombinant P2X3 receptors expressed by human embryonic kidney cells apparently do not show PKC-dependent phosphorylation (Brown and Yule, 2007), other studies have proposed constitutive threonine phosphorylation (Egan et al., 2004) of P2X3 receptors, a property demonstrated in the present investigation with native receptors of sensory neurons and powerfully regulated by NGF levels.

Our study indicated that 24 h administration of anti-NGF antibodies primarily decreased threonine phosphorylation of the P2X3 receptor, a phenomenon accompanied by smaller-amplitude currents with slower recovery. Our experiments also showed that acute application of NGF not only led to the recovery of receptor operation depressed by the previous antibody treatment, but it also promptly increased phosphorylation of P2X3 subunits.

Furthermore, application of NGF per se to naive neurons could upregulate the amplitude of currents mediated by P2X3 receptors and increase the P2X3 subunit phosphorylation state. Globally, our results suggest that, in the present experimental conditions, although P2X3 receptors were constitutively phosphorylated to generate typical current responses to agonist application, their operation could be readily modulated by extracellular levels of NGF. We propose that, because the P2X3 receptor displays a conserved threonine residue in the intracellular N-terminal domain that is part of a protein kinase C consensus site (Mager et al., 2004), blocking NGF might have tilted the balance between protein kinases and phosphatases (Paukert et al., 2001). This process would thus promote a dephosphorylated receptor form generating smaller responses with delayed recovery from desensitization.

Anti-NGF treatment changed subunit composition of P2X receptors

An increase in heteromeric P2X2/3 receptors after anti-NGF antibody application was supported by the demonstration of enhanced membrane expression of P2X2 subunits in association with P2X3 receptors. This novel observation was associated with a change in the kinetics of the α,β-meATP-evoked currents with an increased residual component at the end of the agonist application. In absolute terms, such a residual current was, nevertheless, rather small (<10-fold) when compared with the peak current. It seems, therefore, unlikely that, on TG neurons, the residual current, despite its enhanced size, could be a major contributor to neuronal excitability or nociception. In keeping with this view, we found, with Ca2+ imaging and in vivo animal tests, that neuronal excitability and pain-related behavior dependent on P2X3 receptor activity was reduced after anti-NGF treatment. We propose that, in the present experimental model, the major signal to evoke voltage-dependent Ca2+ transients was the peak current attributable to P2X3 receptor activation. Although heteromeric P2X2/3 receptors have been hypothesized to be involved in mediating chronic pain (North, 2004; Burnstock, 2006), their actual contribution to persistent nociceptive signaling is not fully understood. Our results suggest that the functional impact of heteromeric P2X2/3 receptors may depend not only on the ratio between homomeric and heteromeric channels but also on subunit isoforms.

In accordance with this view, immunoprecipitation experiments indicated that the enhanced expression of P2X2/3 receptors induced by anti-NGF application included the P2X2e splicing variant recently cloned (Koshimizu et al., 2006) and found to confer reduced receptor function (Koshimizu and Tsujimoto, 2006), which would be a contributor to lower neuronal excitability. Conversely, PCR and immunocytochemistry data suggest that P2X2 subunits were insensitive to NGF excess, perhaps because this subunit is already fully regulated by endogenous NGF levels and does not change during culturing conditions (Simonetti et al., 2006).

These observations globally indicate a high level of adaptation of the sensory neuron phenotype, whereby subunit specificity and function can be changed without transcribing a different gene.

Perturbing NGF does not change the action of the algogen CGRP

NGF upregulates synthesis and release of several peptides, including CGRP from peripheral nerve terminals (Heppenstall and Lewin, 2000). We found, on TG neurons, strong P2X3 receptor potentiation after CGRP treatment (Fabbretti et al., 2006). Nevertheless, the effects of adding NGF or NGF neutralization on P2X3 receptor-mediated currents was not mediated by NGF-induced CGRP released because of its insensitivity to the CGRP receptor antagonist BIBN4096BS (Olesen et al., 2004). Furthermore, the high segregation of CGRP and TrkA receptors among different TG neurons made unlikely an action of NGF on CGRP-reactive neurons. This condition allowed observing upregulation of CGRP-evoked P2X3 receptor currents despite the anti-NGF treatment.

Functional implications

Systemic administration of anti-NGF neutralizing antibodies prevents behavioral sensitization, upregulation of neuropeptides, and inflammation-induced expression of the immediate early gene c-fos in dorsal horn nociceptive neurons (Woolf et al., 1994). In accordance with these data, administration of an anti-TrkA antibody is analgesic in a Formalin pain test on mice (Ugolini et al., 2007). In the present study, we observed that anti-NGF treatment prevented trigeminal pain induced by α,β-meATP, even if the trigeminal pain model used in the present investigation is, of course, based on activation of complex mechanisms, of which P2X receptor activity is one contributor. Because NGF levels are primarily increased in migraneurs (Sarchielli et al., 2001), our results support the notion that anti-NGF may represent one analgesic approach to chronically relapsing, severe headache (Saldanha et al., 1999). Future studies are needed to establish whether combined treatments against multiple pain receptors (TRPV1, acid-sensing ion channel, and neurokinins) including P2X3 receptors will be clinically useful and the most appropriate choice of agents for synergy of analgesic effect.

Footnotes

This work was supported by Telethon Grant GGP 04037 and a Fondo per gli Investimenti della Ricerca di Base (Ministero dell'Istruzione, dell'Università, e della Ricerca) grant. We are grateful to Dr. Silvia Dominissini (Unità Malattie Metaboliche, Istituto di Ricovero e Cura a Carattere Scientifico Burlo Garofolo di Trieste, Trieste, Italy) for her help with sequencing experiments. We also thank Boehringer Ingelheim (Ingelheim, Germany) for the generous gift of BIBN4096BS. We are greatly indebted to Dr. Marco Stebel (University of Trieste, Trieste, Italy) for his invaluable help with the behavioral experiments.

Correspondence should be addressed to Andrea Nistri,
Neurobiology Sector, International School for Advanced Studies, Via Beirut 4, 34014 Trieste, Italy.nistri{at}sissa.it